Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 102, Issue 4, Pages 1222-1227Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0406871102
Keywords
pineal; circadian; cAMP; kinase
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The nocturnal increase in circulating melatonin in vertebrates is regulated by the activity of arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme in the melatonin pathway (serotonin --> N-acetylserotonin --> melatonin). Large changes in activity are linked to cyclic AMP-dependent protein kinase-mediated phosphorylation of AANAT T31. Phosphorylation of T31 promotes binding of AANAT to the dimeric 14-3-3 protein, which activates AANAT by increasing arylalkylamine affinity. In the current study, a putative second AANAT cyclic AMP-dependent protein kinase phosphorylation site, S205, was found to be approximate to55% phosphorylated at night, when T31 is approximate to40% phosphorylated. These findings indicate that ovine AANAT is dual-phosphorylated. Moreover, light exposure at night decreases T31 and S205 phosphorylation, consistent with a regulatory role of both sites. AANAT peptides containing either T31 or S205 associate with 114-3-3zeta in a phosphorylation-dependent manner; binding through phosphorylated (p)T31 is stronger than that through pS205, consistent with the location of only pT31 in a mode I binding motif, one of two recognized high-affinity 14-3-3-binding motifs AANAT protein binds to 114-3-3zeta through pT31 or pS205. Two-site binding lowers the K-m for arylalkylamine substrate to approximate to30 muM. In contrast, single-site pS205 binding increases the K-m to approximate to1,200 muM. Accordingly, the switch from dual to single pS205 binding of AANAT to 14-3-3 changes the K-m for substrates by approximate to40-fold. pS205 seems to be part of a previously unrecognized 14-3-3-binding motif-pS/pT (X1-2)-COOH, referred to here as mode III.
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